Vasoactive intestinal polypeptide (VIP) is a widely distributed peptide hormone which mediates a variety of physiological responses including gastrointestinal secretion, relaxation of gastrointestinal, vascular and respiratory smooth muscle, lipolysis in adipocytes, pituitary hormone secretion, and excitation and hyperthermia after injection into the central nervous system (Snedecor and Cochran, Statistical Methods (Ames, Iowa: ISU Press, pp. 508-509 (1967)); Said in Gut Hormones (Bloom and Polak (eds.) 2nd ed., pp. 379-384, New York: Churchill-Livingston, Inc. (1981)). VIP is synthesized as a preprohormone composed of 170 amino acid residues (Cuttitta, et al., J. Clin. Endo. Met., 67:576-583 (1988)). VIP, a 28 amino acid peptide with an amidated C-terminal, results from posttranslational processing (Said & Mutt, Science, 69:1217-1218 (1970)). The VIP peptide has been shown to contain at least two functional regions, a region involved in receptor specific binding and a region involved in biological activity (Gozes and Brenneman, Molecular Neurobiology, 3:201-236 (1989)).
Another biological function of VIP is as a modulatory agent in the central nervous system (CNS) and periphery (Said & Mutt, Science, 69:1217-1218 (1970)). In the rat brain, VIP elevates cAMP levels and stimulates adenylate cyclase in the cortex, striatum, hypothalamus, hippocampus, thalamus, and midbrain (Deschodt-Lanckman, et al., FEBS Lett., 83:76-80 (1977); Etgen and Browning, J. Neurosci., 3:2487-2493; Kerwin, et al., J. Pharm. Pharmacol., 32:561-566 (1980); Quick, et al., Biochem. Pharmacol., 27: 2209-2213 (1978)). Further, VIP fulfills several criteria for a neurotransmitter mediating penile erection. It is present in nerve fibers innervating cavernous smooth muscle and blood vessels and is elevated during erection (Ottesen, et al., Br. Med. J., 288:9 (1984); Dixon, et al., J. Endocrinol., 100:249 (1984)). Injection of exogenous VIP induces erection in man (Ottesen, et al., Br. Med. J., 288:9 (1984)) and penile levels have been shown to be decreased in impotent men (Gu, et al., Lancet, 2:315 (1984)). Since VIP appears to be important in erection formation (Anderson, et al., J. Physiol., 350:209 (1984)), its administration has been found to be helpful in relieving penile dysfunction (See, e.g., Gozes, et al., Endocrinology, 125(4):2945-2949; U.S. Pat. No. 5,147,855 and U.S. Pat. No. 5,217,953).
VIP is also biologically active in the mammalian lung and has been found to be colocalized to cholinergic neurons in the lung (Shimosegawa, et al., Reg. Peptides, 2:181 (1989)). Endogenous VIP is present in nerves supplying airway smooth muscle as well as glands and in pulmonary vessels within the normal adult lung (Ley, et al., Cell Tissue Res., 220:238 (1981)). VIP functions in the lung as a bronchodilator and relaxes pulmonary vascular smooth muscles (Diamond, et al., Am. Rev. Respir. Dis., 128:827-832 (1983); Greenburg, et al., Thorax, 40:715 (1985); Morice, et al., Lancet, 1:457-458 (1984)). VIP has been found to be deficit in the airways of patients with bronchial asthma (Lebacq-Verheyden, et al., J. Cell. Biochem., 36:85-96 (1988)).
The actions caused by VIP may be mediated by specific receptors. VIP receptors were initially detected in the CNS using brain homogenates (Robberecht, et al., Eur. J. Biochem., 90:147-154 (1978)) and, more recently, autoradiographic studies have localized the receptors to discrete brain areas such as the cerebral cortex, striatum, supraoptic nucleus of the hypothalamus, dentate gyms, pinneal and area postrema (Besson, et al., Peptides, 5:339-340 (1984); DeSouza, et al., Neurosci. Lett., 56:113-120 (1985); Shaffer and Moody, Peptides, 7:283-288 (1986)). VIP receptors have also been characterized in liver membranes (Bataille, et al., Endocrinology, 95:713-721 (1974)) and pancreatic acinar cells (Christophe, et al., J. Biol. Chem., 251:4629-4634 (1976)).
The biological actions of VIP in the lung may also be mediated by VIP receptors which have been detected in binding assays using plasma membranes derived from the rat, mouse, guinea pig, and human lung (Christophe, et al., Peptides, 2:253-258 (1981); Dickinson, et al., Peptides 7:791-800 (1986); Robberecht, et al., Peptides, 4:241-250 (1982)). Using in vitro autoradiographic techniques and lung slices, VIP receptors have been localized to the alveoli and epithelium of the rat lung and pulmonary artery smooth muscle and alveolar walls of the human lung (Leroux, et al., Endocrinology, 114:1506-1512 (1984); Leys, et al., FEBS Lett., 199:198-202 (1984)). The lung VIP receptors were characterized using cross-linking techniques and found to have an apparent molecular weight of 67 KDa (Lebacq-Verheyden, et al., Mol. Cell. Biol., 8:3129-3135 (1988)). Additionally, it has been demonstrated that VIP positively regulates adenylate cyclase activity in the lung (Oilerenshaw, et al., N. Engl. J. Med., 320:1244-1248 (1989)).
Recently, it was determined that VIP receptors are present in the malignant lung (Shaffer, et al., Peptides, 8:1101-1106 (1987)). Lung cancer is a serious public health problem which kills approximately 150,000 people in the United States annually (Minna, et al., in: Cancer: Principles and Practice of Oncology (DeVita, et al. (eds.), pp. 507-599 (1985)). Traditionally lung cancer is treated with chemo and/or radiation therapy, but better survival rates might be possible with the development of new modes of therapy. Lung cancer can be divided into small cell lung cancer (SCLC) which accounts for approximately 25% of the lung cancer cases and non-small cell lung cancer (NSCLC). NSCLC can be further subdivided into adenocarcinoma, large cell carcinoma and squamous cell carcinoma each of which account for approximately 25% of the lung cancer cases. SCLC uses bombesin/gastrin releasing peptide (BN/GRP) as an autocrine growth factor (Cuttitta, et al., Nature, 316:823-825 (1985)). Thus, SCLC synthesizes and secretes BN/GRP, and BN or GRP bind to cell surface receptors and stimulate the growth of SCLC. Further, NSCLC synthesizes and secretes transforming growth factor alpha (i.e., TGF-alpha) which, in turns, binds to cell surface epidermal growth factor (EGF) receptors and stimulates NSCLC growth (Imanishi, et al., J. Natl. Cancer Inst., 81:220-223 (1989)). In contrast, VIP receptors are present in cells derived from SCLC and the three other major types of lung cancer (all members of NSCLC), large cell carcinoma, squamous cell carcinoma and adenocarcinoma (Shaffer, et al., Peptides, 8:1101-1106 (1987)).
Recently, Gozes, et al. have developed a VIP antagonist that has proven useful for altering the function of the vasoactive intestinal peptide. (See, U.S. Pat. No. 5,217,953 issued to Gozes, et al. (1993)). This VIP antagonist was designed to retain the binding properties of VIP for its receptor, but to lack the amino acid sequence necessary for biological activity which, it is believed, requires, among other factors, a phenylalanine residue at position 6. Amino acids 1-6 of native VIP were therefore replaced by a segment of neurotensin in order to alter the biological activity of native VIP and to change the membrane permeability of the peptide. Three of the six amino acids added in the neurotensin segment are basic. This is in contrast to native VIP which, in this region, contains no basic residues and only one acidic residue. Indeed, the concept that a tetrapeptide with basic amino acids at both ends and a proline residue adjacent to the N-terminal amino acid is essential for high activity on membrane permeability has been proven correct for neurotensin and other peptides as well. As such, the VIP antagonist developed by Gozes, et al. is a hybrid molecule containing an amino acid sequence necessary for VIP receptor binding (i.e., amino acids 7-28 of VIP), and an N-terminal amino acid sequence corresponding to a portion of neurotensin.
Studies have shown that this VIP antagonist effectively antagonizes VIP-associated activity. More particularly, it has been found that this VIP antagonist inhibits the effect of VIP on the sexual behavior of a mammal. (See, e.g., Gozes, et al., Endocrinology, 125(4):2945-2949; and U.S. Pat. No. 5,217,953.) It has also been found that the hybrid VIP antagonist potently inhibits VIP binding (with a higher affinity than VIP itself); attenuates VIP-stimulated cAMP accumulation; and induces neuronal cell death in tissue culture. (See, e.g., Gozes, et al., J. Pharmacol. Exp. Ther., 257(8): 959-966 (1991).) Moreover, it has been found that this VIP antagonist inhibits the growth of VIP receptor bearing tumor cells such as, for example, lung tumor cells (i.e., NSCLC cells). (See, U.S. Pat. No. 5,217,953.)
Although this VIP antagonist effectively antagonizes VIP-associated activity, there still remains a need for VIP antagonists which are more potent than the VIP hybrid antagonist developed by Gozes, et al. and which are capable of discriminating between the various VIP receptors present in cells. The present invention remedies these needs by providing such antagonists.